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α-Mangostin Alleviated HIF-1α-Mediated Angiogenesis in Rats With Adjuvant-Induced Arthritis by Suppressing Aerobic Glycolysis.

ABSTRACT: A previously validated anti-rheumatic compound α-mangostin (MAN) shows significant metabolism regulatory effects. The current study aimed to clarify whether this property contributed to its inhibition on synovial angiogenesis. Male wistar rats with adjuvant-induced arthritis (AIA) were orally treated by MAN for 32 days. Afterwards, biochemical parameters and cytokines in plasma were determined by corresponding kits, and glycometabolism-related metabolites were further accurately quantified by LC-MS method. Anti-angiogenic effects of MAN were preliminarily assessed by joints based-immunohistochemical examination and matrigel plug assay. Obtained results were then validated by experiments in vitro. AIA-caused increase in circulating transforming growth factor beta, interleukin 6, hypoxia inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in blood and local HIF-1α/VEGF expression in joints was abrogated by MAN treatment, and pannus formation within matrigel plugs implanted in AIA rats was inhibited too. Scratch and transwell assays revealed the inhibitory effects of MAN on human umbilical vein endothelial cells (HUVECs) migration. Furthermore, MAN inhibited tubule formation capability of HUVECs and growth potential of rat arterial ring-derived endothelial cells in vitro. Meanwhile, MAN eased oxidative stress, and altered glucose metabolism in vivo. Glycolysis-related metabolites including glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglyceric acid and phosphoenolpyruvic acid in AIA rats were decreased by MAN, while the impaired pyruvate-synthesizing capability of lactate dehydrogenase (LDH) was recovered. Consistently, MAN restored lipopolysaccharide-elicited changes on levels of glucose and LDH in HUVECs culture system, and exerted similar effects with LDH inhibitor stiripentol on glycometabolism and VEGF production as well as tubule formation capability of HUVECs. These evidences show that MAN treatment inhibited aerobic glycolysis in AIA rats, which consequently eased inflammation-related hypoxia, and hampered pathological neovascularization.

SUBMITTER: Jiang TT

PROVIDER: S-EPMC8721667 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Wutou Decoction (WTD), as a classic prescription, has been generally used to treat rheumatoid arthritis (RA) for two thousand years in China. However, the potential protective effects of WTD on rheumatoid arthritis and its possible mechanism have rarely been reported. Purpose: The aim of this study was to explore the possible mechanism of WTD against RA and a promising alternative candidate for RA therapy. Methods: A model of collagen-induced arthritis (CIA) was constructed in rats to assess the therapeutic effects of WTD. Histopathological staining, immunofluorescence, and western blotting of synovial sections were conducted to detect the antiangiogenic effects of WTD. Then, cell viability assays, flow cytometry, scratch healing assays, and invasion assays were conducted to explore the effects of WTD on MH7A human fibroblast-like synoviocyte (FLS) cell proliferation, apoptosis, migration, and invasion in vitro. The ability of WTD to induce blood vessel formation after MH7A cell and human umbilical vein endothelial cell line (HUVEC) coculture with WTD intervention was detected by a tube formation assay. The mechanisms of WTD were screened by network pharmacology and confirmed by in vivo and in vitro experiments. Results: WTD ameliorated the symptoms and synovial pannus hyperplasia of CIA rats. Treatment with WTD inhibited MH7A cell proliferation, migration, and invasion and promoted MH7A apoptosis. WTD could inhibit MH7A cell expression of proangiogenic factors, including VEGF and ANGI, to induce HUVEC tube formation. Furthermore, the PI3K-AKT-mTOR-HIF-1α pathway was enriched as a potential target of WTD for the treatment of RA through network pharmacology enrichment analysis. Finally, it was confirmed in vitro and in vivo that WTD inhibits angiogenesis in RA by interrupting the PI3K-AKT-mTOR-HIF-1α pathway. Conclusion: WTD can inhibit synovial hyperplasia and angiogenesis, presumably by inhibiting the migration and invasion of MH7A cells and blocking the production of proangiogenic effectors in MH7A cells. The possible underlying mechanism by which WTD ameliorates angiogenesis in RA is the PI3K-AKT-mTOR-HIF-1α pathway.

Project description:ObjectivesReprogramming of cellular metabolism is profoundly implicated in tumorigenesis and can be exploited to cancer treatment. Cancer cells are known for their propensity to use glucose-dependent glycolytic pathway instead of mitochondrial oxidative phosphorylation for energy generation even in the presence of oxygen, a phenomenon known as Warburg effect. The type II beta regulatory subunit of protein kinase A (PKA), PRKAR2B, is highly expressed in castration-resistant prostate cancer (CRPC) and contributes to tumour growth and metastasis. However, whether PRKAR2B regulates glucose metabolism in prostate cancer remains largely unknown.Materials and methodsLoss-of-function and gain-of-function studies were used to investigate the regulatory role of PRKAR2B in aerobic glycolysis. Real-time qPCR, Western blotting, luciferase reporter assay and chromatin immunoprecipitation were employed to determine the underlying mechanisms.ResultsPRKAR2B was sufficient to enhance the Warburg effect as demonstrated by glucose consumption, lactate production and extracellular acidification rate. Mechanistically, loss-of-function and gain-of-function studies showed that PRKAR2B was critically involved in the tumour growth of prostate cancer. PRKAR2B was able to increase the expression level of hypoxia-inducible factor 1α (HIF-1α), which is a key mediator of the Warburg effect. Moreover, we uncovered that HIF-1α is a key transcription factor responsible for inducing PRKAR2B expression in prostate cancer. Importantly, inhibition of glycolysis by the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) or replacement of glucose in the culture medium with galactose (which has a much lower rate than glucose entry into glycolysis) largely compromised PRKAR2B-mediated tumour-promoting effect. Similar phenomenon was noticed by genetic silencing of HIF-1α.ConclusionsOur study identified that PRKAR2B-HIF-1α loop enhances the Warburg effect to enable growth advantage in prostate cancer.

Project description:A previously identified anti-rheumatic compound α-mangostin (MAN) possesses notable metabolism regulatory properties. In this study, we investigated the immune implication of MAN-altered fat metabolism on adjuvant-induced arthritis (AIA) in rats. Seven days after AIA induction, the rats received oral treatment of MAN at 50 mg/kg/day for 30 days. Metabolic indicators and basic clinical parameters were evaluated using samples collected on day 20 and 38 since immunization. Expression of nicotinamide phosphoribosyltransferase (NAMPT), sirtuin 1 (SIRT1), peroxisome proliferator activated receptor gamma (PPAR-γ), stearoyl-coa desaturase 1 (SCD-1), toll like receptor 4 (TLR4), prostaglandin-endoperoxide synthase 2 (COX-2), (p)-JNK, (p)-p65 and IL-1β were investigated by either RT-qPCR or immunobloting methods. In in vitro experiments, we treated (pre)-adipocytes with monocytes/macrophages and MAN, and investigated the changes of macrophages brought by pre-adipocytes co-culture. Generally, MAN restored the impaired fat anabolism in AIA rats, indicated by increased fat reservoir, leptin and adiponectin secretion, and PPAR-γ and SCD-1 expression. Meanwhile, it decreased circulating IL-1β and IL-6 levels, restored serological lipid profile changes, and relieved oxidative stresses, demonstrating potent therapeutic effects on AIA. AIA rats-derived monocytes inhibited mRNA PPAR-γ and SCD-1 expression in pre-adipocytes. Contrarily, MAN facilitated adipocyte differentiation in vitro, and increased free fatty acids production. It also significantly increased PPAR-γ and SCD-1 expression, which can be abrogated by PPAR-γ inhibitor T0070907. Similarly, lipopolysaccharide-primed macrophages inhibited PPAR-γ expression in the co-cultured pre-adipocytes, which was reversed by MAN. In the same co-culture system, lipopolysaccharide-induced inflammation was amplified by the co-existence of pre-adipocytes. More secretion of IL-1β and IL-6 and higher levels expression of COX-2, p-JNK, p-p65 and TLR4 were observed in lipopolysaccharide-treated macrophages when co-cultured by pre-adipocytes. The intensified inflammatory situation was eased by MAN. The treatment with pre-adipocytes culture medium achieved similar effects. Medium from lipopolysaccharide-treated adipocytes promoted IL-1β, IL-6 and MCP-1 production in separately cultured macrophages, and COX-2, p-JNK, p-p65 and TLR4 expression were increased at the meantime. MAN treatment on pre-adipocytes impaired these changes. It suggests that fat anabolism in AIA rats was deficient due to increased energy expenditure caused by inflammatory conditions. MAN restored fat metabolism homeostasis by up-regulating PPAR-γ, and reshaped secretion profile of adipocytes.

Project description:BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by symmetric arthritis. Coix Seed Oil (CSO) has been shown to reduce inflammation in collagen induced arthritis (CIA) rats. However, the effect of CSO on synovial angiogenesis in RA is unknown. In this study, we aimed to explore whether CSO could inhibit RA synovial angiogenesis and elucidate the underlying mechanisms.MethodsCIA rat models were established and subjected to different doses of CSO treatments for four weeks in vivo. Arthritis index, paw swelling, and weight were recorded to assess clinical symptoms. Hematoxylin and Eosin staining, Safarnin O fast green staining, Micro-CT, Immunohistochemical, and Immunofluorescence (IF) staining were performed to examined changes in synovial and joint tissues. The serum HIF-1α and VEGF-A levels were evaluated through enzyme-linked immunosorbent assay. Fibroblast-like synoviocytes (FLS) of rats was stimulated with tumor necrosis factor-α (TNF-α) for developing inflammatory model in vitro. Optimal concentrations of CSO and TNF-α for stimulation were measured through Cell Counting Kit-8 test. Wound healing and Transwell migration experiments were employed to determine FLS migratory ability. IF staining was performed to assess HIF-1α nuclear translocation in FLS. Protein levels of SIRT1, HIF-1α, VEGF-A, and CD31 were assessed through Western blot. The isolated aortic rings were induced with recombinant rat VEGF-A 165 (VEGF-A165) to observe the CSO inhibitory impact on angiogenesis ex vivo.ResultsCSO attenuated the progression of arthritis in CIA rats, mitigated histopathological deterioration in synovial and joint tissues, significantly inhibited immature vessels labeled with CD31+/αSMA-, and reduced the micro-vessels in VEGF-A165 induced aortic rings. Moreover, it upregulated SIRT1 protein levels in CIA rats and TNF-α induced FLS, but decreased HIF-1α and VEGF-A protein levels. Furthermore, CSO inhibited the migration ability and HIF-1α nuclear translocation of TNF-α induced FLS. Finally, suppressing SIRT1 levels in TNF-α induced FLS enhanced their migration ability, HIF-1α nuclear translocation, and the protein levels of HIF-1α, VEGF-A, and CD31, whereas the inhibitory effect of CSO on TNF-α induced FLS was severely constrained.ConclusionsThis study indicates that CSO can alleviate synovial angiogenesis through suppressing HIF-1α/VEGF-A signaling pathways via SIRT1 in CIA rats.

Project description:Background and Objective: Glucose fluctuation (GF) has been reported to induce renal injury and diabetic nephropathy (DN). However, the mechanism still remains ambiguous. Mitochondrial energy metabolism, especially aerobic glycolysis, has been a hotspot of DN research for decades. The activation of HIF-1α/miR210/ISCU/FeS axis has provided a new explanation for aerobic glycolysis. Our previous studies indicated quercetin as a potential therapeutic drug for DN. This study aims to evaluate levels of aerobic glycolysis and repressive effect of quercetin via HIF-1α/miR210/ISCU/FeS axis in a cell model of GF. Methods: The mouse glomerular mesangial cells (MCs) were exposed in high or oscillating glucose with or without quercetin treatment. Cell viability was measured by CCK8 assay. Aerobic glycolysis flux was evaluated by lactate acid, pH activity of PFK. Apoptosis level was confirmed by Annexin V-APC/7-AAD double staining and activity of caspase-3. TNF-α and IL-1β were used to evaluate inflammation levels. Results: GF deteriorated inflammation damage and apoptosis injury in MCs, while quercetin could alleviate this GF-triggered cytotoxicity. GF intensified aerobic glycolysis in MCs and quercetin could inhibit this intensification in a dose-dependent manner. Quercetin prevented activities of two FeS-dependent metabolic enzymes, aconitase, and complex I, under GF injury in MCs. The mRNA expression and protein contents of HIF-1α were increased after GF exposure, and these could be alleviated by quercetin treatment. Knockdown of ISCU by siRNA and Up-regulating of miR-210 by mimic could weaken the effects of quercetin that maintained protein levels of ISCU1/2, improved cell viability, relieved inflammation injury, decreased apoptosis, and reduced aerobic glycolysis switch in MCs. Conclusion: Quercetin antagonizes GF-induced renal injury by suppressing aerobic glycolysis via HIF-1α/miR-210/ISCU/FeS pathway in MCs cell model. Our findings contribute to a new insight into understanding the mechanism of GF-induced renal injury and protective effects of quercetin.

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α-Mangostin Alleviated HIF-1α-Mediated Angiogenesis in Rats With Adjuvant-Induced Arthritis by Suppressing Aerobic Glycolysis.

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